The present invention relates to the preservation of biological tissue using microinjection of intracellular protective agents containing sugar to preserve cells by freezing and/or drying.
In recent years, chemotherapy and radiation therapy of patients with cancer has been increasingly successful and sustained remissions have been achieved. However, the chronic side effects of these therapies to the reproductive systems of long-term survivors is of particular concern. These effects for women include depletion of ovarian germ cells and sterility. Due to the potential loss of future fertility of those exposed to cancer therapy, a need for oocyte banking has developed. Oocyte freezing, when combined with in vitro fertilization, may be beneficial to women desiring future fertility who are anticipating loss of gonadal function from extirpative therapy, radiation, or chemotherapy. Oocyte freezing may also provide a possible alternative to human embryo freezing, thus avoiding many of the legal and ethical problems encountered in embryo freezing.
The first successful cryopreservation of human embryos was achieved in 1983 and embryo freezing is now a routine procedure. In contrast, very limited success has been reported with cryopreservation of human oocytes. Only five successful pregnancies have been reported with more than 1500 cryopreserved oocytes. Therefore, the current methods of freezing are still considered experimental and novel approaches are needed to overcome the difficulty encountered by cryopreservation of the human oocyte.
Traditional cryopreservation techniques include penetrating cryoprotectants at concentrations of 1 to 2M with, for example, dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, followed by a slow freezing rate (0.3 to 0.5xc2x0 C./min). Typically, oocytes are damaged due to long-term exposure to deleterious freezing conditions, including excessive dehydration and high electrolyte concentrations. An alternative approach, called vitrification (i.e. formation of glassy material without crystallization of ice, uses high concentrations of cryoprotectant mixtures (6 to 8M) followed by rapid cooling in order to avoid the lethal effects of freezing on oocytes.
Though an attractive alternative, vitrification procedures suffer from the toxic and osmotic effects of high cryoprotectant concentration on sensitive cells. Neither of these two approaches (slow freeze-thaw and rapid vitrification) has resulted in a reliably successful outcome for cryopreservation of human oocytes. Thus, there is a need for a reliable technique for human oocyte storage. In order to provide the preservation of mammalian cells necessary for application of living cells as a therapeutic tool in clinical medical care, new protocols for preserving living nucleated cells using low levels of non-toxic preservation agents and having simple procedures applicable to a variety of cells must be developed.
The purpose of the present invention is to allow the storage of living cells in a dormant state and the subsequent recovery of the cells to an active state. This method involves microinjecting into the cytoplasm of a cell a protective agent that is substantially non-permeating with respect to mammalian cell membranes and that maintains the viability of the cell such that it can be stored in a temporarily dormant state and substantially restored to an active state. The microinjected cell is subjected to conditions that cause it to enter a dormant state and is stored in this dormant state. The stored cell can be subsequently restored to an active state. This method has the advantage of allowing any mammalian cell to be stored until it is needed under conditions that cause minimal, if any, adverse side-effects in the cell.
Thus, the invention, in some embodiments, provides a method for preserving living cells that begins with microinjecting a protective agent containing an effective sugar into the cell, preferably an oocyte. Other preferred cells that may be preserved include differentiated cells, such as epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells, T-cells, erythrocytes, macrophages, monocytes, fibroblasts, or muscle cells; and undifferentiated cells, such as embryonic, mesenchymal, or adult stem cells. In one preferred embodiment, the differentiated cells remain differentiated after they are recovered from a frozen or dried state, and the undifferentiated cells remain undifferentiated after they are recovered. The cells can be haploid (DNA content of n; where xe2x80x9cnxe2x80x9d is the number of chromosomes found in the normal haploid chromosomes set of a mammal of a particular genus or species), diploid (2n), or tetraploid (4n). Other cells include those from the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, or uterus. The cells may be from a human or non-human mammal, such as a monkey, ape, cow, sheep, big-horn sheep, goat, buffalo, antelope, oxen, horse, donkey, mule, deer, elk, caribou, water buffalo, camel, llama, alpaca, rabbit, pig, mouse, rat, guinea pig, hamster, dog, or cat.
The method of the invention may advantageously use low levels, less than or equal to about 6, 5, 4, 3, 2, or 1 M, or even less than about 0.4 M of preservation agent, and may use a sugar alone as the preservation agent, or sugar in combination with a conventional cryoprotectant, or in combination with other intracellular sugars or extracellular sugars. More preferably, the cytoplasmic concentration of the sugars is less than 0.3, 0.2, 0.1, 0.05, or 0.01 M after microinjection and before freezing or drying of the cell. The extracellular concentration of the sugars is preferably less than 0.3, 0.2, 0.1, 0.05, or 0.01 M after dilution into a liquid medium containing the cell. If the cell is grown on a solid support, such as an agar plate, the concentration of the sugars in the preservation agent that is contacted with the cell is preferably less than 0.3, 0.2, 0.1, 0.05, or 0.01 M. In other preferred embodiments, the final concentration of extracellular sugar in the medium containing the cell is at least 2, 3, 4, 5, or 10 fold greater than the cytoplasmic concentration of intracellular sugar after microinjection and before freezing or drying of the cell. The intracellular and extracellular preservation agents may be the same or different molecular species.
Preferred protective agents include sugars such as monosaccharides, disaccharides, and other oligosaccharides. Preferably, the agent is substantially non-permeable such that at least 50, 60, 70, 80, 90, or 95% of the agent does not migrate across the plasma membrane into or out of the cell, by active or passive diffusion. Preferred sugars have a glass transition temperature of the maximally freeze-concentrated solution (Tgxe2x80x2) that is at least xe2x88x9260, xe2x88x9250, xe2x88x9240, xe2x88x9230, xe2x88x9220, xe2x88x9210, or 0xc2x0 C. Examples of such sugars are those listed in FIG. 8. The Tgxe2x80x2 of other sugars may be routinely determined using standard methods such as those described by Levine and Slade (J. Chem. Soc., Faraday Trans. 1, 84:2619-2633, 1988). The sugar or conventional cryoprotectant with a Tgxe2x80x2 below xe2x88x9250xc2x0 C. can be combined with a sugar with a Tgxe2x80x2 above xe2x88x9250xc2x0 C. such that the resulting mixture has a Tgxe2x80x2 of at least xe2x88x9260, xe2x88x9250, xe2x88x9240, xe2x88x9230, xe2x88x9220, xe2x88x9210, or 0xc2x0 C., and this mixture is used for cryopreservation.
Suitable monosaccarides include those that have an aldehyde group (i.e., aldoses) or a keto group (i.e., ketoses). Monosaccharides may be linear or cyclic, and they may exist in a variety of conformations. Other sugars include those that have been modified (e.g., wherein one or more of the hydroxyl groups are replaced with halogen, alkoxy moieties, aliphatic groups, or are functionalized as ethers, esters, amines, or carboxylic acids). Examples of modified sugars include xcex1- or xcex2-glycosides such as methyl xcex1-D-glucopyranoside or methyl xcex2-D-glucopyranoside; N-glycosylamines; N-glycosides; D-gluconic acid; D-glucosamine; D-galactosamine; and N-acteyl-D-glucosamine. In other preferred embodiments, the preservation agent is an oligosaccharide that includes at least 10, 25, 50, 75, 100, 250, 500, 1000, or more monomers. The oligosaccharide may consist of identical monomers or a combination of different monomers. Other suitable oligosaccharides include hydroxyl ethyl starch, dextran, cellulose, cellobiose, and glucose. Other suitable preservation agents include compounds that contain a sugar moiety and that may be hydrolytically cleaved to produce a sugar. Still other suitable preservation agents include glycoproteins and glycolipids, which preferably have been modified by the addition of 1, 2, 3, 4, 5 or more sugar moieties derived from sugars with a Tgxe2x80x2 of at least xe2x88x9260, xe2x88x9250, xe2x88x9240, xe2x88x9230, xe2x88x9220, xe2x88x9210, or 0xc2x0 C. or with a molecular weight of at least 120 daltons. By xe2x80x9csugar moietyxe2x80x9d is meant a protective sugar that includes a group that can be bonded to another compound. For example, a reactive groupxe2x80x94such as an alcohol, primary amine, or secondary aminexe2x80x94in a sugar can react with a compound, forming a product that includes the sugar moiety.
Another suitable extracellular preservation agent is a lectin or any protein that can non-covalently or covalently bind to a sugar that forms part of a cell-surface glycoprotein or glycolipid. This binding may stabilize the cellular membrane during storage of the cell.
Examples of other cyroprotectants that may be used in the methods of the present invention include sugars, polyols, glycosides, polymers, and soluble proteins with a molecular weight of at least 120 daltons. As illustrated in FIGS. 8 and 9, compounds with higher molecular weights tend to promote glass formation at a higher temperature than that promoted by smaller compounds, allowing the cells to be stored at a higher storage temperature.
After treatment with the microinjected protective agent and, optionally, the external protective agent, the cell is then prepared for storage. In general, the cell may be prepared for storage by freezing and/or drying. Plunge freezing, vacuum drying, air drying, as well as freeze drying techniques may be employed. Typically, oocytes are cooled at a rate of 0.1 to 10xc2x0 C./min, preferably, between 0.3 and 5xc2x0 C./min, and, more preferably, between 0.5 and 2xc2x0 C./min, inclusive. Somatic cells are cooled at a rate between 0.1 and 200xc2x0 C./min, preferably, between 0.5 and 100xc2x0 C./min, and, more preferably, between 1 and 10xc2x0 C./min or 10 and 5xc2x0 C./min, inclusive. The cells are cooled to a final temperature of at least xe2x88x9260, xe2x88x9250, xe2x88x9240, xe2x88x9230, xe2x88x9220, xe2x88x9210, 0, 10, or 20xc2x0 C. (in order of increasing preference). In another preferred embodiment, the preservation agent inhibits or prevents the nucleation or growth of intracellular ice during freezing of the cells.
Extracellular preservation agents may reduce the osmotic shock to the cells that potentially results from the addition of an intracellular preservation agent. Additionally, extracellular preservation agents may stabilize plasma membranes and provide mechanical strength to the cells during freezing or drying.
Once the cell is prepared for storage, it is stored in a manner appropriate to its preparation. Frozen cells can be stored at cryogenic temperatures and dried cells can be dry stored at ambient or other temperatures as appropriate. Recovery of stored cells is geared to the method of their preparation for storage. Dried cells are rehydrated, and frozen cells are thawed. Preferably, at least 25, 35, 50, 60, 70, 80, 90, 95, or 100% of the recovered cells are viable. Cell viability may be measured using any standard assay, such as a xe2x80x9clive/deadxe2x80x9d assay using the green dye calcein-AM to indicate viable cells and the red dye ethidium homodimer to indicate dead cells, according to the manufacturer""s protocol (Molecular Probes, Inc.). In another preferred embodiment, at least 5, 10, 15, 25, 35, 50, 60, 70, 80, 90, or 95% of the recovered oocytes may be fertilized using standard in vitro fertilization techniques (see, for example, Summers et al., Biol. Reprod. 53:431-437, 1995). Preferably, the fertilized oocytes develop into 2-cell stage embryos, 4-cell stage embryos, morula-stage embryos, blastocyst-stage embryos, fetal-stage embryos, or viable offspring.
By xe2x80x9cembryoxe2x80x9d is meant a developing cell mass that has not implanted into the uterine membrane of a maternal host. Hence, the term xe2x80x9cembryoxe2x80x9d may refer to a fertilized oocyte, a pre-blastocyst stage developing cell mass, or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host and prior to formation of a genital ridge. An embryo may represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote; a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.
By xe2x80x9cfetusxe2x80x9d or xe2x80x9cfetalxe2x80x9d is meant a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus may have defining features such as a genital ridge which is easily identified by a person of ordinary skill in the art.
In another aspect, the invention provides a method of culturing mammalian cells in vitro by incubating the cells in a hypertonic media having an osmolarity of greater than 300, 310, 320, 330, 340, 350, 360, 370, 380, or more mosm. Preferably, the media includes one or more of the components listed in FIG. 7 or one or more cryopreservation agents of the present invention. Preferably, the media contains nutrients such as amino acids, sugars, lactate, or pyruvate. This media may be used to culture any mammalian cell, including the preferred cells listed above. In various preferred embodiments, this media is used to culture cells before, during, or after cryopreservation.
The present invention provides a number of advantages related to the cryopreservation of cells. For example, these methods may be generally applied to the preservation of any cell from any mammal. These cells may be stored in a frozen or dried state for any length of time until they are needed. Additionally, these cryopreservation methods involve the use of relatively low concentrations of non-toxic preservation agents that cause minimal, if any, adverse side-effects in the stored cells. Moreover, the preservation agents reduce or eliminate the formation of intracellular ice during freezing which would otherwise damage the cells. If desired, both intracellular and extracellular preservation agents may be used to reduce the osmotic pressure caused by the addition of an intracellular preservation agent to the cells. Extracellular preservation agents may also stabilize plasma membranes and provide mechanical strength to the cells during freezing or drying. Furthermore, the present invention may enable a higher storage temperature (preferably, greater than xe2x88x9260xc2x0 C.) compared to conventional cyroprotectants (typically less than xe2x88x9280xc2x0 C.) due to the high Tgxe2x80x2 of sugars.